Mutagenesis Advance Access originally published online on September 23, 2006
Mutagenesis 2006 21(6):369-374; doi:10.1093/mutage/gel042
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Nitrocompound activation by cell-free extracts of nitroreductase-proficient Salmonella typhimurium strains
Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas Universidad Nacional Autónoma de México. México
A characterization of nitrocompounds activation by cell-free extracts (CFE) of wild-type (AB+), SnrA deficient (B+), Cnr deficient (A+) and SnrA/Cnr deficient (AB–) Salmonella typhimurium strains has been done. The Ames mutagenicity test (S. typhimurium his+ reversion assay) was used, as well as nitroreductase (NR) activity determinations where the decrease in absorbance generated by nitrofurantoin (NFN) reduction and NADP(H) oxidation in the presence of NFN, nitrofurazone (NFZ), metronidazole (MTZ) and 4-nitroquinoline-1-oxide (4NQO) were followed. Different aromatic and heterocyclic compounds were tested for mutagenic activation: 2-nitrofluorene (2-NF); 2,7-dinitrofluorene (2,7-DNF); 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP); 1,6-dinitropyrene (1,6-DNP); and 1,8-dinitropyrene (1,8-DNP). Differential mutagenicity was found with individual cell free extracts, being higher when the wild type or Cnr containing extract was used; nevertheless, depending on the nitrocompound, activation was found when either NR, SnrA or Cnr, were present. In addition, all nitrocompounds were more mutagenic after metabolic activation by CFE of NR proficient strains, although AB– extract still showed activation capacity. On the other hand, NR activity was predominantly catalyzed by wild type CFE followed by A+, B+ and AB– extracts in that order. We can conclude that results from the Ames test indicate that Cnr is the major NR, while NFN and NFZ reductions were predominantly catalyzed by SnrA. The characterization of the residual NR activity detected by the mutagenicity assay and the biochemical determinations in the AB– CFE needs further investigation.
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Introduction |
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Chemical mutagenesis is a complex process that may be strongly influenced by chemical influx/efflux processes, DNA repair, the activity of DNA polymerase at site of DNA lesions and metabolic activation (1
). The later process involves the bacterial enzymes termed nitroreductases (NRs), these enzymes metabolize nitro aromatic compounds into mutagenic products.
In Salmonella enterica subspecies I, serotype Typhimurium (Salmonella typhimurium), SnrA and Cnr NR (2,3) have been recognized based on their homology with Escherichia coli NR NfsA and NfsB (4). SnrA and Cnr share 87 and 88.5% identity with NfsA and NfsB, respectively (2,5). These enzymes have been grouped into oxygen-insensitive (type I) NR (6) and catalyze the pyridine nucleotide-dependent reduction of nitro aromatics to either a hydroxylamine or amine aromatic end product by two-electron steps (7), using NAD(P)H or NADH as a source of reducing equivalents (8,9).
In the S.typhimurium strains employed in the Ames test, NR play an important role in the metabolic activation of several nitrocompounds (10) contained in extracts of diesel and gasoline emissions, fly ash particles, cigarette smoke condensates, home heater emissions and the urban atmosphere (11). The strains employed in the Ames test are not identical in relation to the presence of snrA and cnr genes; hence, differential reduction of nitrosubstituted chemicals would be expected. On the matter, Yamada et al. (12) studied the NR activity and mutagenicity in Ames tester strains hisG46 (TA1535 and TA100), hisD3052 (TA1538 and TA98) and derivative strains with a specifically disrupted cnr gene, and found that these strains were probably not isogenic with respect to their NR activities. Even more, Porwollik et al. (13), confirmed later that hisD3052 (TA1538 and TA98) derivatives, had a snrA gene deletion. These differences in NR activity found among Ames tester strains, allowed Yamada et al. (12) to suggest that the S.typhimurium NR might exhibit dissimilar substrate specificities. Such differences have been found in other tester strains constructed by introducing plasmids that contained snrA and cnr genes (1) supporting the observation that NR might display differential specificity towards nitrocompounds.
Bacterial NR are of toxicological interest due to their participation in nitroimidazole resistance (14–17) xenobiotic biodegradation (18,19) and their utility in prodrug activation (20,21). The study of these enzymes has been focused on their biochemical properties (5,22) their genetic regulation (23,24) and their molecular characteristics (25–27). Results obtained on the NR activity of NfsA and SnrA using NFZ or NFN as substrates, have lead to the conclusion that these enzymes are the major NR in E.coli and S.typhimurium respectively and that NfsB and Cnr are the minor ones (2,5). On the other hand, Cnr deficient Ames Salmonella strains are refractory to the mutagenicity of important environmental nitrocompounds (28) and Cnr overproducing Salmonella strains are very sensitive to the same chemicals (29). These results shows the importance of this enzyme in the mutagenicity of nitrocompounds, but the role of SnrA is less understood.
Our main objective was to examine the nitrocompound activation capacity of cell-free extracts prepared from overnight cultures of the wild-type (SnrA+, Cnr+), SnrA deficient (Cnr+), Cnr deficient (SnrA+) and SnrA/Cnr deficient (SnrA–/Cnr–) strains of S.typhimurium, in an effort to ascertain the role of these NR in nitrocompound mutagenicity.
In the present communication, we report the results of the mutagenicity assays done with several nitrocompounds including nitroaromatics and nitroheterocyclic environmental pollutants.
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Materials and methods |
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Nitrosubstituted chemicals
Test chemicals were obtained commercially (Sigma-Aldrich Co.) and used as received. All nitrocompounds were fresh prepared as required in DMSO: 2-NF (CAS no. 22250-99-3; MW 211.22), 2,7-DNF (CAS no. 5405-53-8; MW 256.22), 1-NP (CAS no. 5522-43-0; MW 247.25), 1,3-DNP (CAS no. 75321-20-9; MW 292.25), 1,6-DNP; (CAS no. 42397-64-8; MW 292.25), 1,8-DNP (CAS no. 42397-65-9; MW 292.25), NFN (CAS no. 67-20-9; MW 238.16), NFZ (CAS no. 59-87-0; MW 198.14), 4-NQO (CAS no. 56-57-5; MW 190.16) and MTZ (CAS no. 443-48-1; MW 171.16).
Bacterial strains and growth conditions
The bacterial strains employed are described in Table I. S.enterica serotype Typhimurium LT2 was used as the standard strain. The S.typhimurium strain YG7132 (hisD3052, gal, 
[chl, uvrB, bio], rfa, cnr– [Kmr], pKM101; Apr, Kmr), was used as the tester strain in the Ames mutagenicity test (12). Bacterial cultures were routinely grown at 37°C on agar plates prepared with Nutrient Broth (Difco®), Agar (1.5%), NaCl (0.5%) and Oxoid nutrient broth #2 (ONB#2; 2.5% w/v; Oxoid Inc.) on a rotating shaker incubator running at 150 r.p.m. Filter sterilized chloramphenicol (20 µg/ml), tetracycline (20 µg/ml) and ampicillin (25 µg/ml), were added to agar plates and media when required.
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snrA and cnr disruption
All the ‘TT’ and ‘TR’ strains in Table I were kindly provided by J.R. Roth, University of California Davis, CA, USA. Transductional crosses were mediated by the phage mutant P22 HT105/1 int-201 (30
). Transductants were single colony purified and made phage-free by streaking on non-selective green indicator plates (31).
Cross-streaking to check phage sensitivity was done with a P22 clear-plaque mutant, H5. Methods for preparing P22 transducing lysates were described previously (32). Insertion of the Tn10d(T-POP2) element in the snrA gene was isolated by transducing the Tn10d(T-POP2) element (from strain TT18797) into a recipient strain TT17437 expressing the Tn10 transposase from plasmid pNK2881 (33,34). Insertion of the Tn10d(T-POP2) element was isolated as Tetracycline resistant (TcR), and 25 µg/ml Nitrofurantoin resistant (NfR) colonies (concentrations 2.5 higher than with the wild type) and were scored by replica printing.
snrA::Tn10d(T-POP2) insertion was transduced into TR7079, generating the RC1378 strain and mapped by PCR. cnr disruption was obtained by transducing the MudCm element from strain TT16528 into a recipient strain TT1704 bearing his deletion. Insertion of the MudCm element was isolated as Chloramphenicol resistant and NfR colonies (at concentrations 2.5 higher than with the wild type) and were scored by replica printing. cnr::MudCm insertion was transduced into TR7079 resulting in the RC1503 strain and mapped by PCR. A double mutant snrA cnr was obtained by transduction, using RC1503 as the recipient and RC1378 as donor, thus generating the RC1517 strain.
Preparation of cell-free extracts
For enzyme and mutagenicity assays, overnight cultures of S.typhimurium strains were grown in ONB#2 supplemented with antibiotics. Cultures were harvested by centrifugation at 10 000x g for 30 min. The cells were washed and suspended in 3 vol. of 50 mM Tris–HCl (pH 7.5), 1 mM dithiothreitol (DTT) (CAS no. 27565-41-9, Sigma-Aldrich). The suspensions were chilled at 4°C, sonically treated with a Microson-Ultrasonic cell disrupter XL-Misonix® for four periods of 30 s each, and centrifuged at 10 000x g for 10 min.
The transparent CFE was suspended in Tris–HCl buffer plus DTT and finally centrifuged at 100 000x g for 60 min at 4°C. Protein determinations were done using the Bradford procedure (Bio-Rad; Richmond, Calif.) with a BSA standard curve.
NR activity
CFE NR activity was determined both, by following the decrease in absorbance at 373 nm (
373 = 21.4 mM–1 cm–1) generated by NFN reduction and the decrease in bsorbance at 340 nm (340 = 6.2 mM–1 cm–1) generated by NAD(P)H oxidation in the presence of nitrocompounds (35) at 32 ± 2°C, using a Camspec M350T (double beam spectrophotometer). The reaction mixture (1 ml) was preincubated at 25°C for 10 min and contained 50 mM Tris–HCl (pH 7.5), 1 mM DTT, 100 µM ß-NAD(P)H or NADH, CFE (50 µg total protein) and 25 µM of each nitrocompound tested: NFN, NFZ, MTZ, 4NQO. Reactions were initiated by the addition of NAD(P)H or NADH solution and monitored for the change in the amounts of substrates every 20 s in the first 4 min, in order to obtain the initial reaction velocity, during which the enzyme reaction was linear. All experiments were done in aerobic conditions.
Mutagenicity assay
The Salmonella mutagenicity plate incorporation test was carried out according to the method described by Maron and Ames (36) using CFE of each S.typhimurium strain as metabolic activation systems, and theYG7132 strain deficient in cnr and snrA, as the tester strain. ONB#2 supplemented with ampicillin and kanamicine (25 µg/ml) was used for an overnight culture of strain YG7132. The suspension mixture (370 µl, total volume), consisting on 240 µM of NAD(P)H and CFE (250 µg total protein), the test compound (in 10 µl dimethylsulfoxide) and 100 µl of YG7132 strain, was sequentially added to a tube containing 2 ml of 45°C warm soft agar, mixed and poured onto a Petri dish containing 40 ml minimal agar (15 mg/ml agar in Vogel-Bonner E medium with 20 mg/ml glucose). After incubation for 2 days in the dark, the revertant colonies (His+) were counted. Each dose of test compounds was selected from previous dose–response curves and tested in triplicate. The mean number of His+ revertants per plate was calculated. The background bacterial lawn was carefully examined for evidence of toxicity. All the experiments were done in duplicate.
Statistics
One-way ANOVA and Tukey tests were employed for comparisons between NR activities obtained in the presence of either NAD(P)H or NADH, and for the comparison of NR activities with different CFE and substrates. The software SALANAL (Salmonella Assay Analysis, v. 1.0, Integrated Laboratory Systems, Research Triangle Park, NC, USA) was used for the evaluation of a positive dose–response (slope at origin, P < 0.05). A difference in the activating capacity of two cell free extracts was considered significant when a factor of 2 existed between their mutagenic potencies.
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Results |
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The S.typhimurium strains constructed as described in Table I, were used after specific tests for genotype verification. Initially, we compared CFE nitroreduction activities using NFN as substrate and NAD(P)H or NADH as cofactors (Figure 1); this was done to assess the NR activity in each CFE using standard biochemical methods. Among the NR containing extracts, the CFE of wild-type strain (AB+) showed the highest activity when NADPH was present in the reaction mixture with NFN as substrate, Cnr deficient CFE (A+) displayed also a clear NR activity and SnrA deficient CFE (B+) showed the lowest activity (Figure 1). When NADH was used as cofactor, there was a 75 and 95% reduction in the activity observed with AB+ and A+ CFE, respectively. No statistical difference was noted with SnrA/Cnr deficient CFE (AB–) and B+ CFE in relation with the use of NAD(P)H or NADH (Figure 1).
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Oxidation of NAD(P)H in the presence of various nitroaromatics as substrates was also used to evaluate the NR activity of the four CFE. The highest activity was obtained by AB+ CFE followed by A+ and B+ CFE with NFZ and NFN as substrates (Figure 2). The A+ CFE was more efficient than B+ in metabolizing NFZ and NFN (P < 0.05); nevertheless, the small difference in activity seen with 4-NQO between A+ or B+ cytosolic fractions had no statistical significance. The NR activity obtained with MTZ as a substrate was the lowest observed with the nitroheterocyclic compounds tested, showing no differences in activity among the cytosolic fractions. Finally, the residual activity of AB– detected with MTZ, NFZ and 4-NQO was undetectable when NFN was used as a substrate (Figure 2). The differences in the NFN reductase activity found with each CFE and NAD(P)H observed in Figures 1 and 2, are due to the different methodologies used to evaluate the enzymatic activity.
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P < 0.05 (versus A+ and AB–). 
P > 0.05 (versus B+ to the same chemical).In this study, S.typhimurium YG7132 was chosen as the tester strain because it is defective in both snrA and cnr genes (12
), thus, the selection of nitrocompounds was restricted to those that predominantly produce frameshift mutations after activation to more reactive metabolites. The dose range of the test compounds to be used in the mutagenicity assays were selected from previous experiments (data not shown) with the nitrocompounds activated by the AB+ CFE. With the selected concentrations for each nitrocompound, the mean number of induced revertants per plate was obtained. In comparison with AB– CFE, all nitrocompounds but 1,6-DNP were activated by SnrA or Cnr NR present in the CFE used for the mutagenicity test. Table II shows the results from the mutagenic dose–response curves and the mutagenic potency of each nitrocompound included in this study. With the exception of 1,3-DNP, all the nitrocompounds tested showed a positive mutagenic dose–response in the absence of any CFE. Furthermore, the addition of AB– CFE increases more than twice the mutagenic potency of 2,7-DNF, 1,3-DNP and 1,6-DNP and more than 1.5 times that of 1-NP and 1,8-DNP obtained without CFE. Nitrofluorenes and 1,3-DNP were more efficiently activated by AB+ CFE followed by B+ CFE. On the other hand, 1-NP and 1,8-DNP resulted in a higher mutagenic potency when B+ CFE was present. The mutagenic potency of the former was reduced 1.5 times with the AB+ CFE. Finally, the mutagenic potency of 1,6-DNP did not depend on any of the CFE's in particular. The lowest mutagenic potency calculated from mutagenicity plates was obtained when no cytosolic fraction was present (control plates).
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Discussion |
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In the present communication, we report the effects of NR proficient and deficient S.typhimurium CFE on the mutagenicity of six nitrocompounds, in an effort to elucidate the role of each NR in the mutagenic activation of these chemicals. Under our laboratory conditions, neither of the NR genes interrupted appears to be essential for bacterial growth.
In E.coli, the oxygen-insensitive (type I) NR activity consist of one major and two minor components. The major component is an NAD(P)H-linked enzyme encoded by nfsA, while minor components, encoded by nfnB and an unidentified gene responsible for the production of a protein described as NR IB2 (16,37), can use both NADH and NAD(P)H as electron donors. This cofactor differentiation is in accordance with our results on the NR activity obtained with the S.typhimurium CFE and NFN as a substrate (Figure 1). The remnant NR activity in AB– CFE (Figure 1) suggests that additional factors may modulate NFN reduction including a NR distinct from NfsA or NfsB but similar to the E.coli IB2 NR.
NFN and NFZ reduction are well documented in the literature, in fact, these nitrocompounds are habitually used as substrates in NR activity measurements (1,5,24,38). In spite of the fact that our experiments were carried out with NFN in aerobic conditions, the different results on NR activity found in A+ and AB+ CFE, are in accordance with Rau and Stolz (39). Using NAD(P)H as a cofactor and NFZ as a substrate in anaerobic conditions, these authors found an increment in cell extract activity with NfsA and wild-type E.coli strains and demonstrated differences between NfsA and NfsB NR activity. Our results also showed differences between A+ and B+ CFE with NFZ as a substrate and demonstrated that the major activity was present in the A+ CFE under aerobic conditions.
Results of the mutagenicity tests suggest that the NR present in S.typhimurium CFE used, participate in the activation of all the nitrocompounds tested. Our results demonstrated that the use of S.typhimurium YG7132, was effective in the detection of reduced nitrocompounds since its deficiency in both NR allowed us to evaluate the contribution of the NR present in each CFE.
The Salmonella tester strains TA1535 and TA1538 employed in the Ames assay are not isogenic considering their NR activity (12,13). S.typhimurium TA1535 carries both SnrA and Cnr NR, while TA1538 carries only Cnr NR. The differences found in the metabolic activation by these strains and their cnr disrupted derivatives, constitute an approximation to the specificity of different NR towards nitrocompounds. Nevertheless, the role of each, SnrA and Cnr, in nitrocompound activation to mutagenic products has not been clarified.
Our results in Table II show that the highest mutagenicity of nitrofluorenes was obtained with AB+ CFE, and that the presence of the two enzymes is necessary for the optimal activation of 2,7-DNF and 2-NF. Evidences for activation of 2-NF by Cnr NR has been provided by experiments using Salmonella strains overproducing Cnr or strains disrupted at the Cnr gene (12,29). In these experiments, the mutagenic potency of 2-NF in the overproducing Cnr strain YG1021 was 20 times higher than that obtained with the wild-type strain YG1020. However, in the present work, the participation of Cnr in the activation of 2-NF is not as clear as that reported by the cited authors; this could be due to differences in enzyme concentration.
The nitroreduction of 1-NP by Cnr leading to mutagenic intermediates detected in the Ames test had also been demonstrated (12,28,29). The mutagenic potency of 1-NP was 24-fold higher in a Cnr overproducing strain compared to that observed with the wild-type strain. Our results confirm these data and afford new insights on the participation of SnrA in 1-NP activation (Table II) taking into account the modest increase in mutagenic potency noted in the presence of this NR.
The first step in dinitropyrene activation is its reduction to arylhydroxylamines, catalyzed by NR and the second step is the O-acelylation or the N-acelylation mediated by NAT/OAT (40). This metabolic pathway is supported by results with OAT-deficient strains which are refractory to the mutagenic action of 1,3- 1,6- and 1,8-DNP (28,41) and with OAT-overproducing strains very sensitive to 1,8-DNP (42). On the other hand, a marked decrease in the mutagenic potency of 1,3-DNP but not of 1,6-DNP or 1,8-DNP was obtained when tested in the deficient Cnr strain TA98NR obtained by classical chemical mutagenesis (43). Results in Table II partially agreed with the data mentioned before. AB+ and B+ CFE's efficiently activated 1,3-DNP and the presence of A+ CFE gave the same mutagenic potency as AB– CFE suggesting a null participation of SnrA in 1,3-DNP activation. 1,6-DNP was equally activated by all the CFE's tested indicating an equal and weak participation of both NR in its reduction. Finally, in comparison with AB– CFE, the mutagenic potency of 1,8-DNP increased 1.7 times when CFE containing Cnr was present, suggesting a modest but clear participation of the Salmonella classical NR in the nitroreduction of 1,8-DNP. The weak effect of AB+ CFE on the activation of 1,8-DNP could be due to an interference of SnrA with this activation under the test conditions.
We can conclude that the S.typhimurium NR, SnrA and Cnr, activate the tested nitrocompounds with varying efficiency. Cnr is the major enzyme involved in nitrocompound activation to mutagens and, although SnrA is also able to activate almost all of them, the reduced metabolites are produced in minor concentrations or they are not as mutagenic as those produced by Cnr. An alternative explanation is that the rate of transcription of Cnr is higher than that of SnrA leading to a lower enzymatic concentration of the SnrA. As far as we know, no other NR besides Cnr and SnrA have been described in S.typhimurium. Nevertheless, our results with AB– CFE in the biochemical and mutagenesis assays, suggest the existence of other enzymatic activity involved in the metabolism of the nitrocompounds tested. The differences between Cnr and SnrA substrate specificity observed from biochemical determinations and the mutagenicity test could be attributed to the accepting electron properties of each substrate, or their different chemical structure. The use of biochemical methods like NAD(P)H oxidation with several nitrocompounds comprising a wide range of redox potential and the use of purified Salmonella NR, are in process in order to understand the differences in enzyme specificity.
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Acknowledgments |
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The authors thank Dra. Regina Montero for critically review of the manuscript, Dr Bernardo Frontana Uribe and Dra. Adela Rodriguez Romero for helpful discussions and technical assistance. This work was supported by PAPPIT UNAM grant N° IN223205.
Conflict of Interest Statement: None declared.
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Notes |
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*To whom correspondence should be addressed. Tel: +52 56229214; Fax: +52 56229182; Email: jjea{at}servidor.unam.mx
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Received on June 20, 2006; revised on August 21, 2006; accepted on August 23, 2006.
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